This invention relates to a peptide immunogen comprising a novel artificial T helper cell epitope (Th) covalently linked to a desired target antigenic site comprising B cell epitopes and optionally a general immune stimulator sequence. The artificial Th epitope imparts to the peptide immunogen the capability to induce strong T helper cell-mediated immune responses and the production of antibodies directed against the xe2x80x9ctarget antigenic site.xe2x80x9d The invention also provides for the advantageous replacement of carrier proteins and pathogen-derived T helper cell sites in established peptide immunogens by the novel artificial T helper cell epitopes for improved immunogenicity.
Many rules have been developed for predicting the amino acid sequences of T cell epitopes. However, because there is no central unifying theory on how or what makes a particular amino acid sequence useful as a T cell epitope, the rules are empirical and are not universally applicable. Being aware of these rules, the novel artificial T helper cell epitopes of the present invention were developed, nevertheless, by empirical research.
The peptide immunogens of the present invention are useful for evoking antibody responses in an immunized host to a desired target antigenic site, including sites taken from pathogenic organisms, and sites taken from normally immunosilent self-antigens and tumor-associated targets. Accordingly, the peptides of the invention are useful in diverse medical and veterinary applications, such as: vaccines to provide protective immunity from infectious disease; immunotherapies for treating disorders resulting from malfunctioning normal physiological processes; immunotherapies for treating cancer and as agents to intervene in normal physiological processes to produce desirable results.
For example, the novel artificial T helper cell epitopes of the present invention provide novel short peptide immunogens that elicit antibodies targeted to luteinizing hormone-releasing hormone (LHRH) and are useful for contraception, control of hormone-dependent tumors, prevention of boar taint, and immunocastration. The novel artificial Th epitopes of the present invention have been found to provoke an immune response when combined with target B cell epitopes of various microorganisms/proteins/peptides. In addition to LHRH, the artificial Th epitopes of the present invention have been found to be useful when linked to other target antigenic sites include somatostatin for growth promotion in farm animals; IgE for treatment of allergy; the CD4 receptor of T helper cells for treatment and prevention of HIV infection and immune disorders and foot-and-mouth disease virus capsid protein for prevention of foot-and-mouth disease.
It is known that most antibody immune responses are cell-mediated, requiring cooperative interaction between antigen-presenting cells, B cells (antibody-producing cells which also function as antigen-presenting cells), and T helper (Th) cells. Consequently, the elicitation of an effective antibody response requires that the B cells recognize the target antigenic site (B cell epitope) of a subject immunogen and the T helper cells recognize a Th epitope. Generally, the T helper epitope on a subject immunogen is different from its B cell epitope(s) (Babbitt et al., Nature, 1985; 317: 359-361). The B cell epitope is a site on the desired target recognized by B cells which in response produce antibodies to the desired target site. It is understood that the natural conformation of the target determines the site to which the antibody directly binds. The T helper cell recognition of proteins is, however, much more complex and less well understood. (Cornette et al., in Methods in Enzymology, vol 178, Academic Press, 1989, pp 611-634).
Under present theories, evocation of a Th cell response requires the T helper cell receptor to recognize not the desired target but a complex on the membrane of the antigen-presenting cell formed between a processed peptide fragment of the target protein and an associated class II major histocompatibility complex (MHC). Thus, peptide processing of the target protein and a three-way recognition is required for the T helper cell response. The three part complex is particularly difficult to define since the critical MHC class II contact residues are variably positioned within different MHC binding peptides (Th epitopes) and these peptides are of variable lengths with different amino acid sequences (Rudensky et al., Nature, 1991; 353:622-627). Furthermore, the MHC class II molecules themselves are highly diverse depending on the genetic make-up of the host. The immune responsiveness to a particular Th epitope is thus in part determined by the MHC genes of the hoist. In fact, it has been shown that certain peptides only bind to the products of particular class II MHC alleles. Thus, it is difficult to identify promiscuous Th epitopes, i.e., those that are reactive across species and across individuals of a single species. It has been found that the reactivity of Th epitopes is different even among individuals of a population.
The multiple and varied factors for each of the component steps of T cell recognition: the appropriate peptide processing by the antigen-processing cell, the presentation of the peptide by a genetically determined class II MHC molecule, and the recognition of the MHC molecule/peptide complex by the receptor on T helper cells have made it difficult to determine the requirements for promiscuous Th epitopes that provide for broad responsiveness (Bianchi et al., EP 0427347; Sinigaglia et al., chapter 6 in Immunological Recognition of Peptides in Medicine and Biology, ed., Zegers et al., CRC Press 1995, pp 79-87).
It is clear that for the induction of antibodies, the immunogen must comprise both the B cell determinant and Th cell determinant(s). Commonly, to increase the immunogenicity of a target, the Th response is provided by coupling the target to a carrier protein. The disadvantages of this technique are many. It is difficult to manufacture well-defined, safe, and effective peptide-carrier protein conjugates for the following reasons:
1. Chemical coupling are random reactions introducing heterogeneity of size and composition, e.g., conjugation with glutataraldehyde (Borras-Cuesta et al., Eur J Immunol, 1987; 17: 1213-1215);
2. the carrier protein introduces a potential for undesirable immune responses such as allergic and autoimmune reactions (Bixler et al., WO 89/06974);
3. the large peptide-carrier protein elicits irrelevant immune responses predominantly misdirected to the carrier protein rather than the target site (Cease et al., Proc Natl Acad Sci USA, 1987; 84: 4249-4253); and
4. the carrier protein also introduces a potential for epitopic suppression in a host which had previously been immunized with an immunogen comprising the same carrier protein. When a host is subsequently immunized with another immunogen wherein the same carrier protein is coupled to a different hapten, the resultant immune response is enhanced for the carrier protein but inhibited for the hapten (Schutze et al., J Immunol, 1985; 135: 2319-2322).
To avoid these risks, it is desirable to replace the carrier proteins. T cell help may be supplied to a target antigen peptide by covalent binding to a well-characterized promiscuous Th determinant. Known promiscuous Th are derived from the potent pathogenic agents such as measles virus F protein (Greenstein et al., J Immunol, 1992; 148: 3970-3977) and hepatitis B virus surface antigen (Partidos et al., J Gen Virol 1991; 72: 1293-1299). The present inventors have shown that many of the known promiscuous Th are effective in potentiating a poorly immunogenic peptide, such as the decapeptide hormone luteinizing hormone-releasing hormone (LHRH) (U.S. Pat. No. 5,759,551). Other chimeric peptides comprising known promiscuous Th epitopes with poorly immunogenic synthetic peptides to generate potent immunogens have been developed (Borras-Cuesta et al., 1987). Well-designed promiscuous Th/B cell epitope chimeric peptides are capable of eliciting Th responses with resultant antibody responses targeted to the B cell site in most members of a genetically diverse population (U.S. Pat. No. 5,759,551).
A review of the known promiscuous Th epitopes shows that they range in size from approximately 15 to 50 amino acid residues (U.S. Pat. No. 5,759,551) and often share common structural features with specific landmark sequences. For example, a common feature is the presence of amphipathic helices. These are alpha-helical structures with hydrophobic amino acid residues dominating one face of the helix and charged and polar resides dominating the surrounding faces (Cease et al., 1987). Known promiscuous Th epitopes also frequently contain additional primary amino acid patterns such as a charged residue, -Gly-, followed by two to three hydrophobic residues, followed in turn by a charged or polar residue (Rothbard and Taylor, EMBO J, 1988; 7:93-101). Th epitopes with this pattern are called Rothbard sequences. It has also been found that promiscuous Th epitopes often obey the 1, 4, 5, 8 rule, where a positively charged residue is followed by hydrophobic residues at the fourth, fifth and eighth positions, consistent with an amphipathic helix having positions 1, 4, 5 and 8 located on the same face. This pattern of hydrophobic and charged and polar amino acids may be repeated within a single Th epitope (Partidos et al., J Gen Virol, 1991; 72:1293-99). Most, if not all, of the known promiscuous T cell epitopes contain at least one of the periodicities described above.
Promiscuous Th epitopes derived from pathogens include the hepatitis B surface and core antigen helper T cell epitopes (HBsAg Th and HBc Th), the pertussis toxin helper T cell epitopes (PT Th), the tetanus toxin helper T cell epitopes (TT Th),the measles virus F protein helper T cell epitopes (MVF Th), the Chlamydia trachomatis major outer membrane protein helper T cell epitopes (CT Th), the diphtheria toxin helper T cell epitopes (DT Th), the Plasmodium falciparum circumsporozoite helper T cell epitopes (PF Th), the Schistosoma mansoni triose phosphate isomerase helper T cell epitopes (SM Th), and the Escherichia coli TraT helper T cell epitopes (TraT Th). The sequences of these pathogen-derived Th epitopes can be found in U.S. Pat. No. 5,759,551 as SEQ ID NOS:2-9 and 42-52 therein, incorporated herein by reference; in Stagg et al., Immunology, 1993; 79;1-9; and in Ferrari et al., J Clin Invest, 1991; 88: 214-222, also incorporated by reference
The use of such pathogen-derived sites for the immuno-potentiation of peptide B cell sites for application to LHRH has been described in U.S. Pat. No. 5,759,551, for HIV in Greenstein et al. (1992), for malaria in EP 0 427,347, for rotavirus in Borras-Cuesta et al. (1987), and for measles in Partidos et al. (1991).
Useful Th epitopes may also include combinatorial Th epitopes. In Wang et al. (WO 95/11998), a particular class of combinatorial Th epitopes, a xe2x80x9cStructured Synthetic Antigen Libraryxe2x80x9d (SSAL) was described. Th SSAL epitopes comprise a multitude of Th epitopes with amino acid sequences organized around a structural framework of invariant residues with substitutions at specific positions. The sequences of the SSAL are determined by retaining relatively invariant residues while varying other residues to provide recognition of the diverse MHC restriction elements. This may be accomplished by aligning the primary amino acid sequence of a promiscuous Th, selecting and retaining as the skeletal framework the residues responsible for the unique structure of the Th peptide, and varying the remaining residues in accordance with known MHC restriction elements. Lists of the invariant and variable positions with the preferred amino acids of MHC restriction elements are available to obtain MHC-binding motifs. These may be consulted in-designing SSAL Th epitopes (Meister et al., Vaccine, 1995; 13:581-591).
The members of the SSAL may be produced simultaneously in a single solid-phase peptide synthesis in tandem with the targeted B cell epitope and other sequences. The Th epitope library sequences are designed to maintain the structural motifs of a promiscuous Th epitope and at the same time, accommodate reactivity to a wider range of haplotypes. For example, the degenerate Th epitope xe2x80x9cSSAL1 TH1xe2x80x9d (WO 95/11998), was modeled after a promiscuous epitope taken from the F protein of the measles virus (Partidos et al., 1991 ). SSAL1 TH1 was designed to be used in tandem with a target antigen, LHRH. Like the measles epitope from which it was derived, SSAL1 TH1 was designed to follow the Rothbard sequence and the 1, 4, 5, 8 rules: and ISA Mixture of Four Peptides
A charged residue Glu or Asp is added at position 1 to increase the charge surrounding the hydrophobic face of the Th. The hydrophobic face of the amphipathic helix is then maintained by hydrophobic residues at 2, 5, 8, 9, 10, 13 and 16. Positions at 2, 5, 8, 9, 10, and 13 are varied to provide a facade with the capability of binding to a wide range of MHC restriction elements. The net effect of the SSAL feature is to enlarge the range of immune responsiveness of the artificial Th (WO 95/11998).
Other attempts have been made to design xe2x80x9cidealizedxe2x80x9d artificial Th epitopesxe2x80x9d incorporating all of the properties and features of known promiscuous Th epitopes. Several peptide immunogens comprising these artificial promiscuous Th epitopes, including those in the form of SSAL, have also been constructed. The artificial Th sites have been combined with peptide sequences taken from self-antigens and foreign antigens to provide enhanced antibody responses to site specific targets (WO 95/11998) that have been described as highly effective. Such peptide immunogens are preferred for providing effective and safe antibody responses, and for their immunopotency, arising from a broadly reactive responsiveness imparted by the idealized promiscuous Th site described.
The present invention provides an immunogenic peptide composition comprising a promiscuous artificial T helper cell epitope linked to a synthetic peptide B ell epitope or xe2x80x9ctarget antigenic sitexe2x80x9d. The immunogenic peptide comprise an artificial T helper cell (Th) epitopes and a target antigenic site containing B cell epitopes and, optionally, a general immune stimulator sequence. The presence of an artificial Th epitope in the immunogenic peptide impart thereto a capability to induce a by strong T helper cell-mediated immune response with the production of a high level of antibodies directed against the xe2x80x9ctarget antigenic site.xe2x80x9d The present invention further provides for the advantageous replacement of carrier proteins and pathogen-derived T helper cell sites in established peptide immunogens with artificial T helper cell epitopes designed specifically to improve their immunogenicity. The novel short peptide immunogens with the artificial Th epitpoes of the present invention elicit a high level of antibodies targeted to luteinizing hormone-releasing hormone (LHRH) useful for contraception, the control of hormone-dependent tumors, the prevention of boar taint, and immunocastration.
The artificial Th epitopes were developed empirically, mindful of the known rules for predicting promiscuous T cell epitopes. In the absence of a unifying theory explaining the mechanism of Th epitopes, these xe2x80x9cpredictivexe2x80x9d rules serve merely as guidelines for designing effective artificial Th epitopes. The artificial Th epitopes of the present invention have been found to be useful when linked to target antigenic sites and optionally with a immunostimulatory sequence. The immunogenic peptides of the present invention may be represented by the formulae:
xe2x80x83(A)n-(Target antigenic site)-(B)o-(Th)m-X
or
(A)n-(Th)m-(B)o-(Target antigenic site)-X
or
(Target antigenic site)-(B)o-(Th)m-(A)n-X
or
(Th)m-(B)o-(Target antigenic site)-(A)n-X
wherein:
A is an amino acid or a general immunostimulatory sequence, e.g., the invasin domain (Inv) SEQ ID NO:78 [any others?] where n is more than one, the individual A""s may be the same or different;
B is selected from the group consisting of amino acids, xe2x80x94NHCH(X)CH2SCH2COxe2x80x94, xe2x80x94NHCH(X)CH2SCH2CO(xcex5-N)Lys-, xe2x80x94NHCH(X)CH2S-succinimidyl(xcex5-N)Lys-, and xe2x80x94NHCH(X)CH2S-(succinimidyl)-;
Th is an artificial helper T cell epitope selected from the group consisting of SEQ ID NOS: 6-22, 31-35 and 105 and an analog or segment thereof;
xe2x80x9cTarget antigenic sitexe2x80x9d is a desired B cell epitope, a peptide hapten, or an immunologically reactive analog thereof;
X is an amino acid xcex1-COOH or xe2x80x94CONH2;
n is from 1 to about 10;
m is from 1 to about 4; and
o is from 0 to about 10.
An example of a peptide hapten as a target antigenic site is LHRH (SEQ ID NO: 77.
The compositions of the present invention comprise peptides capable of evoking antibody responses in an immunized host to a desired target antigenic site. The target antigenic site may be derived from pathogenic organisms and normally immunosilent self-antigens and tumor-associated targets such as LHRH.
Accordingly, the compositions of the present invention are useful in many diverse medical and veterinary applications. These include vaccines to provide protective immunity from infectious disease, immunotherapies for the treatment of disorders resulting from the malfunction of normal physiological processes, immunotherapies for the treatment of cancer, and agents to desirably intervene in and modify normal physiological processes.
In addition to LHRH, other useful targets include: somatostatin for growth promotion in farm animals; IgE for treatment of allergy; the CD4 receptor of T helper cells for treatment and prevention of HIV infection and immune disorders; and foot-and-mouth disease virus capsid protein as a vaccine for the prevention of foot-and-mouth disease.